Determining the influence of 3D chromatin structure on human evolution

确定 3D 染色质结构对人类进化的影响

• 批准号: 10271272
• 负责人: Lana Harshman
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-16 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10271272
• 关键词: 3-Dimensional; Affect; Binding; Binding Sites; CCCTC-binding factor; CRISPR/Cas technology; Categories; Cell Line; Cell Survival; Cells; Chromatin; Chromatin Loop; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Consensus; DNA; Data; Data Set; Ensure; Eukaryota; Evolution; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genome; Genomic Segment; Genomics; Haploid Cells; Haploidy; Hi-C; Hominidae; Human; Human Cell Line; Human Genome; Hylobates Genus; Individual; Lead; Lentivirus; Libraries; Limb structure; Location; Mammals; Measures; Methods; Modernization; Molecular Conformation; Morphology; Neighborhoods; Organism; Pan Genus; Phenotype; Play; Positioning Attribute; Prevalence; Recording of previous events; Regulatory Element; Repression; Research Personnel; Role; Scanning; Site; Structure; Time; Untranslated RNA; Work; base; computerized tools; driving force; experimental study; fascinate; genomic data; in silico; nonhuman primate; novel; organizational structure; three dimensional structure; trait; transcription factor; trend

PROJECT SUMMARY/ABSTRACT The importance of 3D genome structure on the evolution of human specific gene regulation, phenotypes, and speciation remains largely unknown. Here, I plan to characterize in a high- throughput manner the impact of topologically associated domains (TADs), which are genomic neighborhoods of self-interacting DNA, and CCCTC binding factor (CTCF) binding motifs, which help facilitate DNA looping, on cell viability and evolution. I hypothesize that a subset of TAD boundaries are critical for maintaining important gene regulation throughout evolution and that derived CTCF binding sites in the human lineage may have led to gene expression changes. I will interrogate this hypothesis by first performing a high-throughput CRISPR-deletion screen in which I will delete a set of over 300 evolutionarily conserved and human specific TAD boundaries in a human haploid cell line to determine if any of these boundaries are essential for cell viability (Aim 1). Second, I will identify human specific CTCF binding motifs that are gained or lost in humans or have orientation changes by comparing genome sequences from human, chimpanzee, and two extinct archaic hominids: Neanderthal and Denisovan. I will further interrogate the importance of a subset of these novel CTCF sites by CRISPR/Cas9 editing human cells to mimic that of an extinct genome and phenotype them for changes in gene expression changes and chromatin conformation. It will then be possible to determine if these changes caused differences in gene regulation and genome looping (Aim 2). Combined, this proposal will take a crucial step in further understanding how 3D chromatin structure can affect phenotypes especially as it relates to human evolution.

项目概要/摘要 3D基因组结构对人类特定基因调控进化的重要性， 表型和物种形成仍然很大程度上未知。在这里，我打算以一个高的角度来描述—— 吞吐量方式影响拓扑相关域 (TAD)，这些域是基因组 自相互作用 DNA 邻域和 CCCTC 结合因子 (CTCF) 结合基序， 有助于促进 DNA 循环、细胞活力和进化。我假设 TAD 的一个子集 边界对于在整个进化过程中维持重要的基因调控至关重要 并且人类谱系中衍生的 CTCF 结合位点可能导致基因 表情发生变化。我将通过首先执行高通量来质疑这个假设 CRISPR 删除屏幕，其中我将删除一组 300 多个进化上保守且 人类单倍体细胞系中的人类特异性 TAD 边界，以确定是否存在以下任何一个 边界对于细胞活力至关重要（目标 1）。其次，我将识别人类特异性CTCF 通过比较在人类中获得或丢失或具有方向变化的结合基序 人类、黑猩猩和两种已灭绝的古原始人类的基因组序列：尼安德特人 和丹尼索万。我将进一步询问这些新的 CTCF 站点子集的重要性 通过 CRISPR/Cas9 编辑人类细胞以模仿已灭绝的基因组并对其进行表型分析 用于基因表达变化和染色质构象的变化。然后就可以 确定这些变化是否导致基因调控和基因组循环的差异（目标 2）。 结合起来，该提案将在进一步了解 3D 染色质如何 结构可以影响表型，特别是当它与人类进化相关时。